5 CYTOCHEMISTRY OF SIALOGLYCOCONJUGATES AND
role in the preservation of skin integrity. However, precise analyses at the cytological level of sialic acids and antimicrobial substances in the carpal glands have not been available until now.
In the present study, therefore, the glandular acini of the porcine carpal glands were subjected to cytochemical analyses of sialoglycoconjugates and the antimicrobial peptide group of β-defensins. The purpose was to determine which cell compartments are engaged in the secretory pathway of these moieties. The ultrastructural data obtained may be indispensable to understand the general biological function of these glands.
5. 2. Materials and Methods
All experiments were performed in accordance with the guidelines for the care and use of laboratory animals at the Institute of Experimental Animal Science, College of Bioresource Sciences, Nihon University. Five male miniature pigs (potbelly, 1-2 years, 40-50 kg) were deeply anesthetized and then exsanguinated from the common carotid arteries. After bloodletting, the carpal glands were removed surgically.
For general structural observation by electron microscopy, the specimens were fixed in 2.5% glutaraldehyde (GA) solution in 0.1 M phosphate-buffered solution (PB) (pH 7.4) for 2 h at 4˚C. The materials were post-fixed in 2% osmium tetroxide solution for 2 h and embedded in Epon 812 (Luft, 1961). From these tissue blocks,
ultrathin sections were cut using an ultramicrotome, mounted on copper grids and stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963).
For glycoconjugate cytochemistry, the tissue specimens were fixed in a mixture of 4% paraformaldehyde (PFA) and 0.5% GA in 0.1 M PB (pH 7.4) for 2 h at 4˚C, and embedded in LR-White resin (Newman et al., 1983). From the LR-White-embedded blocks, ultrathin sections were cut as detailed above and placed on nylon or nickel grids. The sections on nylon grids were reacted for a periodic acid-thiocarbohydrazide-silver proteinate-physical development procedure (PA-TCH-SP-PD) (Yamada, 1993). For cytochemical identification of glycogen in the cytoplasm, enzyme digestion with α-amylase (from Bacillus subtilis, Seikagaku Kogyo Co., Tokyo, Japan, 1 mg/ml, at 37°C for 4 h) (Casselman, 1959) was carried out on some sections prior to the PA-TCH-SP-PD procedure. For the lectin cytochemistry, the nickel grid-mounted sections were incubated with biotinylated lectins at concentrations of 10-20 μg/ml in 0.05 M phosphate-buffered saline (PBS) (pH 7.2) for 24 h at 4˚C (Roth, 1983, 1996), following preincubation with 1% bovine serum albumin (BSA) (Sigma, MO, USA) in PBS. The lectins used were Sambucus sieboldiana agglutinin (SSA) and Maackia amurensis agglutinin (MAM) (Seikagaku Kogyo Co.). Their specific sugar residues and inhibitory sugars are listed in Table 10 (for lectin specificities, see Danguy, 1995). After rinsing with PBS, these sections were incubated with 15 nm colloidal gold-labeled streptavidin (British Biocell
International, Cardiff, UK) at a dilution of 1:20 in PBS for 1 h at room temperature (Roth, 1983). They were then subjected to counterstaining with uranyl acetate and lead citrate. For specificity controls of the cytochemical lectin procedures, ultrathin sections were incubated with the respective lectin solutions to which 0.01 M of an appropriate competing sugar was added or incubated, substituting unconjugated lectins for biotinylated lectins.
For immunocytochemistry, ultrathin sections cut from the LR-White-embedded blocks were also placed on nickel grids.
Following pretreatment with a solution containing 5% donkey serum albumin (DSA) (Jackson ImmunoResearch Labs., PA, USA) in 0.01 M PBS (pH 7.3) to prevent non-specific reactions, the sections were incubated with cross-reacting primary antibodies for human β-defensin 2 (dilution 1:600, polyclonal, anti-human, from rabbit) (Biolog, Kronshagen, Germany) for 24 h at 4˚C. After rinsing with PBS, these ultrathin sections were incubated with biotinylated secondary antibody (anti-rabbit immunoglobulins, from donkey) (Jackson ImmunoResearch Labs.) at a dilution of 1:500 in PBS containing 5% DSA for 60 min, and then with 15 nm colloidal gold-labeled streptavidin at a dilution of 1:20 in PBS for 60 min.
They were counterstained with uranyl acetate and lead citrate.
Controls for the immunocytochemical procedures were performed by incubation with PBS without primary antibodies or by replacement of the primary antibodies with normal rabbit immunoglobulin diluted to the same extent as the specific
antibodies.
Table 10. The lectins used and their sugar-binding specificities and inhibitory sugars Inhibitory sugar α2-6sialyllactose α2-3sialyllactose Sugar-binding specificity Siaα2-6Gal/GalNAc Siaα2-3Galβ1-4GlcNAc
Lectins Sambucussieboldiana agglutinin Maackiaamurensisagglutinin
SSA MAM
5. 3. Results
Observation of the porcine carpal glands by electron microscopy revealed that the glandular acini consist of dark cells and clear cells with associated myoepithelial cells. Additionally, we classified the dark cells as type I and type II dark cells, depending on their morphological characteristics. The secretory cells and myoepithelial cells rested on a basal lamina (Fig. 24a). Both types of dark cell were equipped with a well-developed Golgi apparatus, rough-surfaced endoplasmic reticulum and a large number of secretory granules. Regarding the morphological difference between the two types of dark cell, the type I dark cells contained relatively large secretory granules. On the other hand, the secretory granules of the type II dark cells were smaller than those of type I dark cells (Fig. 24b). The plasma membrane of the clear cells facing the glandular lumen projected into well-developed microvilli. The cytoplasm of the clear cells showed lower electron density, and secretory granules could not be found. Nevertheless, parts of the Golgi apparatus were frequently observed in the circumnuclear region. Abundant smooth-surfaced endoplasmic reticulum and lysosomes were detectable in this cell type. In addition, large numbers of glycogen particles were distributed in the cytoplasm (Fig. 24c, d). Throughout the cytoplasm of the secretory cells, especially the apical region of the clear cells, many mitochondria of varying morphology were scattered among the ultrastructures mentioned above (Fig. 24c).
In the dark cells, the prominent features exhibiting a positive PA-TCH-SP-PD reaction were the secretory granules and cisternae of the Golgi apparatus (Fig. 25a-c). Although the secretory granules of the type I dark cells showed a weak to moderate positive reaction, a distinct positive reaction was observed in those of the type II dark cells (Fig. 25a, b). On the other hand, in the clear cells, glycogen particles, cisternae of the Golgi apparatus and lysosomes exhibited positive reactions (Fig. 25a, b). Other PA-TCH-SP-PD-reactive structures were the surface coat of the plasma membrane of the dark and clear cells. Digestion with α-amylase abolished the PA-TCH-SP-PD-reactive glycogen particles in the cytoplasm.
The SSA-gold procedure demonstrated reactive particles as concentrated in the secretory granules of the type I and type II dark cells (Fig. 26a). In both types of dark cell, the Golgi apparatus also showed a positive reaction (Fig. 26b). With regard to the MAM-gold technique, in the secretory cells, the free surface coat of the plasma membrane showed a positive reaction. The secretory granules of the type I dark cells were seen to react almost negatively, while distinctly concentrated reactive particles were observed in those of the type II dark cells (Fig. 27a). Furthermore, in the type II dark cells, several gold particles were associated with the Golgi apparatus after incubation with MAM (Fig. 27b). Concerning these lectin cytochemical procedures, the clear cells were almost negative. In control sections reacted for lectin cytochemical staining by addition of the appropriate inhibitory sugars to the respective lectin solutions
and substitution of the unconjugated lectins, positive reaction staining of all the formerly reactive ultrastructures was greatly suppressed or abolished.
As for the immunocytochemical methods using the primary antibody to human β-defensin 2, a few gold particles were detectable in the secretory granules of the type II dark cells.
However, those of type I dark cells were very weak or negative, and the clear cells also reacted negatively (Fig. 28a). In addition to these features, reactive particles could be observed in the cisternae of the Golgi apparatus of the type II dark cells (Fig. 28b). With regard to the control ultrathin sections stained by immunocytochemical procedures by incubation with PBS without the primary antibodies or by replacement of the primary antibody with normal rabbit immunoglobulin, no glandular structures exhibited any positive reactions.
Fig. 24 General ultrastructure of the porcine carpal glands stained with uranyl acetate and lead citrate. a) The secretory portion consists of type I dark cells, type II dark cells and clear cells. ×5,000, BL: basal lamina, CC:
clear cell, DC1: type I dark cell, DC2: type II dark cell, L: lumen, Me:
myoepithelial cell, Mi: mitochondrion, Mv: microvilli, N: nucleus, SG:
secretory granule.
Fig. 24 General ultrastructure of the porcine carpal glands stained with uranyl acetate and lead citrate. b) Higher magnification of the dark cells in the glandular acini. ×15,500, DC1: type I dark cell, DC2: type II dark cell, Go: Golgi apparatus, Mi: mitochondrion, N: nucleus, PM: plasma membrane, SG: secretory granule.
Fig. 24 General ultrastructure of the porcine carpal glands stained with uranyl acetate and lead citrate. c) Higher magnification of the clear cells in the glandular acini. ×12,000, Go: Golgi apparatus, L: lumen, Ly: lysosome, Mi: mitochondrion, Mv: microvilli, N: nucleus, sER: smooth-surfaced endoplasmic reticulum, arrows: glycogen particles.
Fig. 24 General ultrastructure of the porcine carpal glands stained with uranyl acetate and lead citrate. d) Part of supranuclear cytoplasm of the clear cells. ×16,500, Go: Golgi apparatus, Ly: lysosome, Mi: mitochondrion, N: nucleus, sER: smooth-surfaced endoplasmic reticulum, arrows:
glycogen particles.
Fig. 25 Cytochemical PA-TCH-SP-PD staining of the carpal glands. a) Ultrastructures exhibit positive reactions. ×8,000, CC: clear cell, DC1:
type I dark cell, DC2: type II dark cell, L: lumen, Mv: microvilli, N:
nucleus, PM: plasma membrane, SG: secretory granule, arrows:
glycogen particles.
Fig. 25 Cytochemical PA-TCH-SP-PD staining of the carpal glands. b) The luminal side of the glandular acini. ×8,000, CC: clear cell, DC1: type I dark cell, DC2: type II dark cell, L: lumen, Mv: microvilli, N: nucleus, PM: plasma membrane, SG: secretory granule, arrows: glycogen particles.
Fig. 25 Cytochemical PA-TCH-SP-PD staining of the carpal glands. c) Part of the supranuclear cytoplasm in the type II dark cell. ×18,500, Go: Golgi apparatus, PM: plasma membrane, SG: secretory granule, asterisks:
immature secretory granules.
Fig. 26 SSA-gold staining of the carpal glands. a) Part of the apical cytoplasm of the dark cells. Reaction in the secretory granules of the type I and type II dark cells. ×16,500, DC1: type I dark cell, DC2: type II dark cell, L: lumen, Mi: mitochondrion, PM: plasma membrane, SG: secretory granule.
Fig. 26 SSA-gold staining of the carpal glands. b) Part of the supranuclear cytoplasm of the type II dark cell. Reactions in the cisternae of the Golgi apparatus and secretory granules. ×21,000, Go: Golgi apparatus, N: nucleus, PM: plasma membrane, SG: secretory granule.
Fig. 27 MAM-gold staining of the carpal glands. a) Part of the luminal side of the glandular acini. Distinctly positive reactions are detectable in the secretory granules of the type II dark cells and in the free surface coat of the plasma membrane of the secretory cells. ×16,000, CC: clear cell, DC1: type I dark cell, DC2: type II dark cell, L: lumen, Mv: microvilli, cell, PM: plasma membrane, SG: secretory granule.
Fig. 27 MAM-gold staining of the carpal glands. b) Part of the supranuclear cytoplasm of the type II dark cell. Reactions in the cisternae of the Golgi apparatus and secretory granules. ×21,500, Go: Golgi apparatus, Mi: mitochondrion, N: nucleus, SG: secretory granule.
Fig. 28 Immunogold labeling for β-defensin 2 of the carpal glands. a) Part of the luminal side of the glandular acini. A clearly positive reaction is detectable in the secretory granules of the type II dark cell. ×12,000, CC:
clear cell, DC1: type I dark cell, DC2: type II dark cell, L: lumen, Mv:
microvilli, N: nucleus, PM: plasma membrane, SG: secretory granule.
Fig. 28 Immunogold labeling for β-defensin 2 of the carpal glands. b) Part of the supranuclear cytoplasm of the type II dark cell. Reactions in the cisternae of the Golgi apparatus and secretory granules. ×18,000, Go:
Golgi apparatus, Mi: mitochondrion, PM: plasma membrane, SG:
secretory granule, asterisks: immature secretory granules.
5. 4. Discussion
The glandular acini of mammalian eccrine glands consist of two different cell types, dark cells and clear cells. The secretory granules of the dark cells contain glycoproteins, whereas the clear cells are involved in producing the hypertonic or isotonic precursor fluid of sweat (Kurosumi et al., 1984). In several mammalian species including humans, previous studies disclosed the presence of glycoconjugates with various saccharide residues in the dark cells (Tsukise et al., 1983; Meyer and Bartels, 1989; Meyer and Tsukise, 1995; Sames et al., 1999; Stumpf and Welsch, 2002; Yasui et al., 2004, 2005a). The distinction of cell types of among dark cells has not been reported previously. However, our investigation demonstrated some slight morphological differences among the dark cells. Furthermore, the two types of dark cell were also distinguished by the results of the PA-TCH-SP-PD procedure, which detects the presence of glycoconjugates with vicinal diol groupings (Yamada, 1993). On the other hand, an abundant smooth-surfaced endoplasmic reticulum was observed in the clear cells compared with that in the eccrine glands in porcine snout skin (Fukui et al., 2012a) and in the foot pads of carnivores (Meyer and Bartels, 1989; Yasui et al., 2005b). The presence of smooth-surfaced endoplasmic reticulum is an important feature of clear cells, suggesting their possible involvement in glycogen storage and in ion concentration and transport (see Kurosumi et al., 1982; Gargiulo et al., 1989). Additionally, distinct numbers of glycogen particles and many mitochondria, as
detectable in the clear cells, seem to be related to high energy demands during their production (Meyer and Bartels, 1989; Sato et al., 1989).
From the results of the lectin cytochemical procedures, in the porcine carpal glands, sialic acid residues linked to α2-6Gal/GalNAc were localized in the secretory granules and cisternae of the Golgi apparatus in both types of dark cell. In contrast, the presence of sialoglycoconjugates with a Siaα2-3Galβ1-4GlcNAc sequence and the antimicrobial peptide group of β-defensins was mainly confined to those of the type II dark cells. These results correspond with sialoglycoconjugate histochemical and immunohistochemical observations of the carpal glands by light microscopy (Fukui et al., 2012b). Such features differ from those of the eccrine glands in other mammals (Meyer and Bartels, 1989; Sames et al., 1999; Stumpf and Welsch, 2002; Yasui et al., 2004, 2005a, 2010). It is considered that the morphological and morphochemical characteristics of the dark cells may depend on the different functional stages or maturation stages.
Sialic acids are widely known to occupy the terminal position of oligosaccharide chains in a variety of glycoconjugates and are considered to perform a key role as the most versatile function modulators in cell biology and pathology. They are involved in binding transport of ions, stabilizing the conformation of proteins and enhancing the viscosity of mucins, owing to their negative charge. Sialic acids function not only as biological masks that are antirecognition agents that act by shielding recognition sites, such as
for macrophages, enzymes or galectins, but also as recognition sites for various molecules, for example, hormones, lectins, antibodies and inorganic cations, as well as for microorganisms (Schauer, 2004;
2009; Varki and Schauer, 2009). Furthermore, sialoderivatives may play an important role in the general defense against pathogenic agents via variation in the types of linkage and acceptor sugars and their degree of acetylation because, for example, influenza viruses differ in their ability to recognize Sia-Gal linkages depending on the animal hosts (Suzuki, 2005; Parillo et al., 2009).
In mammals, β-defensins are produced by phagocytes and various epithelial cells, and are often present at high concentration (Ganz, 2003, 2004). Defensins have a role in the innate immune response of the skin against microbial invaders. They have the capability of insertion into cell membranes, depending upon their electrical charge. Consequently, most defensins cause the destruction of microorganisms, that is, bacteria, fungi and viruses (Bos et al., 2001; Yang et al., 2001; Ganz, 2003, 2004). The antimicrobial peptide group of β-defensins is distributed in the eccrine glands of several mammals (Ali et al., 2001; Stumpf and Welsch, 2002; Stumpf et al., 2004; Yasui et al., 2010). A previous study showed the identification and first characterization of eleven porcine β-defensins and the expression of seven porcine β-defensins in skin tissues (Sang et al., 2006).
In conclusion, our cytochemical analyses give a detailed description of the distribution of sialoglycoconjugates and
β-defensin. Additionally, these results confirm that the dark cells of porcine carpal glands can be classified into type I and type II dark cells, and the Siaα2-3Galβ1-4GlcNAc sequence and β-defensin are produced by the type II dark cells (Fukui et al., 2012b). These secretory components may have an important role in effective protection of the skin surface of the carpal region against environmental pathogens. Therefore, the carpal glands are closely involved in effective defense for the preservation of skin integrity, besides their function as odoriferous glands.
5. 5. Summary
The functional properties of sialic acids appear to be manifold.
Additionally, antimicrobial peptides are effective molecules for innate immunity. In humans and other mammals, defensins are one of the main antimicrobial peptide families. The present study demonstrated the localization of sialoglycoconjugates and β-defensin in the glandular acini of porcine carpal glands using cytochemical methods for electron microscopy. The secretory portion of the carpal glands was found to be composed of two types of dark cells and clear cells. Sialoglycoconjugates that terminated in Siaα2-6Gal/GalNAc were present predominantly in the secretory granules and Golgi apparatus of both types of dark cell. The presence of sialic acid residues linked to α2-3Galβ1-4GlcNAc was mainly restricted to the type II dark cells. The distributional pattern
of β-defensin was consistent with that of the Siaα2-3Galβ1-4GlcNAc sequence. Their presence and secretion are suggestive of protective effects of these secretory products at the skin surface of the carpal region.